The methane producing bacteria are an unusual group of organisms found in the human intestinal system, ruminants, lake bottoms and other anaerobic environments. Little is known about their physiology and biochemistry, or their effects on their host. Some of their unusual cofactors, e.g., a 5-deazaflavin, have potential toxicity. The objective of this research project is to examine the intermediates and enzymes involved in the reaction: 4H2 + CO2 directional CH4 + 2H2O, which is carried out by the methane producing bacteria, generally as their sole energy yielding process. One methanogenic genus can also catalize the reaction: 4CH3OH directional 3CH4 + CO2 2H2O. The enzyme hydrogenase, which provides electrons for the production of CH4, will be studied in several catalytic and kinetic ways in order to determine its rate limiting step, kinetic mechanism and active site structure. Assays at different pH values and temperatures, and in the presence of various solvents will be used to probve the properties of the catalytic site. Carbon intermediates will be examined using approaches with both 13C and 14C labeled CO2 and CH3OH. Short term labveling of cells and extracts with 14C will be analyzed using HPLC, TLE and TLC; similar experiments with 13C will be analyzed by NMR. Inhibitors known to act in the methanogenic pathway will be examined as possible tools in dissecting the reaction sequence. Previous short term labeling studies have revealed an unknown carbon carrying intermediate (YFC) probably a pterin. Its enzymatic conversion to an unlabeled carbon "acceptor" molecule will be examined, as will its ability to donate 14C-label to other putative intermediates. The enzymatic interconversion of this compound (YFC) will also be studied spectrally.